An important new and rapidly growing area of research in biology involves deciphering the "regulatory code" used in developmental programs of higher organisms. The broad objectives of the proposed research are to gain further knowledge on the gene regulatory networks (GRNs) that regulate cell-type specification and differentiation during embryo development. The development of sea urchin embryo pigment cells, a cell-type of mesodermal origin, will be used as the research model. A Delta-Notch signaling is known to be necessary for the differential specification of presumptive mesodermal (secondary mesenchyme cells, SMCs) and endodermal territories. Specifically, the analysis of the cis-regulatory regions of a differentiation gene will be used as a bottomup approach to uncover the structure of the GRN regulating the specification and differentiation processes of pigment cells. The differentiation gene studied is a polyketide synthase (SpPks) required for the biosynthesis of the pigment echinochrome. The cis-regulatory investigation of this gene will serve as a model to characterize in the future the co-regulation of a putative pigment cell gene battery. A comparative genomic approach among different sea urchin species will be used to identify putative cis-regulatory modules. The availability of the complete S. purpuratus genome sequence, of BAC library sequences from other sea urchin species, and of various computational tools will most definitely accelerate the process of identifying proximal and distal cis-regulatory modules. Subsequently, the putative cis-regulatory sequences will be experimentally tested by fusing them to a reporter gene (green fluorescent protein). GFP expression will be monitored throughout development in order to characterize the regulatory functionality of the promoter elements. The functionality of DNA-binding sites within the cis-regulatory elements identified will be tested by site-directed mutagenesis and gel shift assays. A complementary approach proposed is the study of SpPks transcriptional regulation by identifying the transcription factors that bind to the DNA of its promoter. This will be achieved by using the yeast one-hybrid (Y1H) system. Currently the sea urchin gene regulatory network for endo-mesoderm specification is the most extensively characterized developmental GRN in any organism. Nevertheless, the genetic circuits downstream of endomesoderm specification leading to differentiated cell-types are poorly understood. The results of this project will generate important knowledge on the control mechanisms of GRNs during the later phase of development. The integration of the knowledge of the genetic basis of cell differentiation with earlier cell specification will provide further understanding of the developmental genetic programs at the system level, greatly increasing our ability to prevent and treat human diseases in a novel way.